This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tremlett, C. H. Articles by Woodford, N. Search for Related Content PubMed PubMed Citation Articles by Tremlett, C. H. Articles by Woodford, N.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, March 2001, p. 996-997, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.996-997.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

LETTERS TO THE EDITOR

Two Structurally Distinct VanA Resistance Elements at Different Locations in a Glycopeptide-Resistant Strain of Enterococcus faecalis

	LETTER

Glycopeptide-resistant enterococci (GRE) can carry more than one VanA element (3, 5), but we are unaware of previous evidence for separate transfer of such elements. Enterococcus faecalis JS3B was examined as one of 46 GRE isolated from fecal screens of a hematology patient at Addenbrooke's Hospital, Cambridge, United Kingdom (7). Overlapping PCR (1) showed that this strain contained a group H element (11). VanA resistance appeared to be associated with a 35-MDa plasmid, but Palepou et al. (6) suggested that group H elements reside on the chromosome. Strain JS3B was therefore investigated for carriage of multiple VanA elements.

Transfer of vancomycin resistance was performed by cross-streak conjugation (10). The recipient strains were E. faecalis JH2-2 and E. faecium GE-1, which are resistant to fusidic acid and rifampin and lack pheromone response genes prgA and prgB (see below). After incubation at 37°C for 72 h, the transconjugants were selected on brain heart infusion agar (Oxoid) containing 100 µg of rifampin per ml, 25 µg of fusidic acid per ml, and 10 µg of vancomycin per ml. Plates were incubated at 37°C and examined daily for 5 days. Eight to 12 individual colonies were subcultured onto Columbia horse blood agar. The colonial characteristics of selected transconjugants were noted, and plasmid profiles were examined following alkaline lysis and agarose gel electrophoresis (8).

Five transconjugants, representing each combination of plasmid profile, hemolysis reaction, and colonial morphology, were subjected to overlapping PCR (1, 11). Plasmids were resubjected to alkaline lysis, followed by Southern blotting onto a nylon membrane, and hybridized with a digoxigenin-labeled vanA-specific probe (2). Strain JS3B and the five transconjugants were also examined for the conserved pheromone response genes prgA (which encodes entry exclusion protein) and prgB (which encodes aggregation substance protein) with previously described PCR primers (4) and the same cycling conditions as for overlapping PCR.

Three transconjugants represented the E. faecalis JH2-2 host and contained group U elements. Two transconjugants represented E. faecium GE-1; one contained a group U element, whereas the other had a group H element (Table 1). Each of the four transconjugants with group U elements possessed a plasmid of ca. 35 MDa that hybridized with the vanA probe. These four transconjugants and JS3B all possessed prgA and prgB. The transconjugant with the group H element did not possess detectable plasmids, and the vanA probe hybridized only with residual chromosomal DNA. However, it is possible that the group H element may be carried on a large conjugative plasmid not recovered by the alkaline lysis technique employed here (9). The transconjugant with the group H element lacked pheromone response genes prgA or prgB, suggesting that transfer of its VanA resistance element did not depend on a pheromone-responsive conjugative plasmid.

View this table: [in this window] [in a new window]   TABLE 1.   Characteristics of transconjugants derived from E. faecalis JS3B

In conclusion, strain JS3B contained two distinct VanA elements, a group U element carried on a pheromone-responsive plasmid of ca. 35 MDa and a group H element that appeared to be chromosomal. In overlapping PCR, the group H element, which yielded multiple amplicons, masked the group U element, which yielded fewer amplicons (11). These results stress the importance of using multiple molecular techniques to identify the structure and location of VanA elements in GRE.

	ACKNOWLEDGMENTS

This work was supported by a grant from the PHLS Small Scientific Initiative Fund.

	FOOTNOTES

^* Phone: (44)-1223-257035 Fax: (44)-1223-242775

	REFERENCES

1.	Arthur, M., C. Molinas, F. Depardieu, and P. Courvalin. 1993. Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J. Bacteriol. 175:117-127[Abstract/Free Full Text].
2.	Garaizer, J., M. E. Kauffman, and T. L. Pitt. 1991. Comparison of ribotyping with conventional methods for type identification of Enterobacter cloacae. J. Clin. Microbiol. 29:1303-1307[Abstract/Free Full Text].
3.	Handwerger, S., J. Skoble, L. F. Discotto, and M. J. Pucci. 1995. Heterogeneity of the vanA gene cluster in clinical isolates of enterococci from the northeastern United States. Antimicrob. Agents Chemother. 39:362-368[Abstract/Free Full Text].
4.	Heaton, M. P., and S. Handwerger. 1995. Conjugative mobilization of a vancomycin resistance plasmid by a putative Enterococcus faecium sex pheromone response plasmid. Microb. Drug Res. 1:177-183.
5.	Jensen, L. B., P. Ahrens, L. Dons, R. N. Jones, A. M. Hammerum, and F. M. Aarestrup. 1998. Molecular analysis of Tn1546 in Enterococcus faecium isolated from animals and humans. J. Clin. Microbiol. 36:437-442[Abstract/Free Full Text].
6.	Palepou, M. I., A. A. Adeyibi, C. H. Tremlett, L. B. Jensen, and N. Woodford. 1998. Molecular analysis of diverse elements mediating VanA glycopeptide resistance in enterococci. J. Antimicrob. Chemother. 42:605-612[Abstract/Free Full Text].
7.	Tremlett, C. H., D. F. J. Brown, and N. Woodford. 1999. Variation in structure and location of VanA glycopeptide resistance elements among enterococci from a single patient. J. Clin. Microbiol. 37:818-820[Abstract/Free Full Text].
8.	Uttley, A. H. C., C. H. Collins, R. C. George, J. Naidoo, N. Woodford, A. P. Johnson, C. H. Collins, D. Morrison, A. J. Gilfillan, L. E. Fitch, and J. Heptonstall. 1989. High-level vancomycin-resistant enterococci causing hospital infections. Epidemiol. Infect. 103:173-181[Medline].
9.	Werner, G., I. Klare, and W. Witte. 1999. Large conjugative plasmids in vancomycin-resistant Enterococcus faecium. J. Clin. Microbiol. 37:2383-2384[Free Full Text].
10.	Woodford, N., D. Morrison, A. P. Johnson, A. C. Bateman, J. G. M. Hastings, T. S. J. Elliott, and B. Cookson. 1995. Plasmid-mediated vanB glycopeptide resistance in enterococci. Microb. Drug Res. 1:235-240.
11.	Woodford, N., A.-M. A. Adebiyi, M.-F. I. Palepou, and B. D. Cookson. 1998. Diversity of VanA glycopeptide resistance elements in enterococci from humans and nonhuman sources. Antimicrob. Agents Chemother. 42:502-508[Abstract/Free Full Text].

					Catherine H. Tremlett* Derek F. J. Brown Clinical Microbiology and Public Health Laboratory Addenbrooke's Hospital Cambridge CB2 2QW United Kingdom
					Marie-France I. Palepou Neil Woodford Antibiotic Resistance Monitoring and Reference   Laboratory Central Health Public Laboratory London NW9 5HT United Kingdom

This article has been cited by other articles:

• Novais, C., Coque, T. M., Sousa, J. C., Baquero, F., Peixe, L. (2004). Local Genetic Patterns within a Vancomycin-Resistant Enterococcus faecalis Clone Isolated in Three Hospitals in Portugal. Antimicrob. Agents Chemother. 48: 3613-3617 [Abstract] [Full Text]  

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tremlett, C. H. Articles by Woodford, N. Search for Related Content PubMed PubMed Citation Articles by Tremlett, C. H. Articles by Woodford, N.